Team:Lethbridge/Results

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Project Results

THEOPHYLLINE APTAZYME IN VITRO:

As the theophylline aptazyme used in our project had been previously utilized in vivo, exhibiting cleavage in response to theophylline, we first wanted to observe if it was viable in vitro. In order to do this, our group first generated a PCR product of the full length aptazyme and used it in an in vitro transcription.

As illustrated, our transcription of the aptazyme was successful, however we noted the presence of a significant amount of cleaved aptazyme even in the absence of theophylline. Initially, we hypothesized that this may be due to the temperature at which our in vitro transcription was performed, as elevated temperature may impair proper folding of the structure resulting in premature cleavage. We therefore attempted in vitro transcription at room temperature as well as 37 degrees.

As illustrated, we were able to generate observable quantities of full length aptazyme. This is the first time this has been exhibited in vitro, which is a significant accomplishment. However, we wanted to obtain pure, full-length aptazyme. As such, we tested our in vitro transcription across a wide variety of temperatures, timepoints, and reactant concentrations.

Upon concluding that complete purification of full-length aptazyme was potentially not possible using these in vitro conditions, we wanted to see if addition of theophylline would still result in further cleavage. Using concentrations of theophylline ranging from 100nM to 1mM, cleavage of the aptazyme was analyzed.

Unexpectedly, cleavage of the aptazyme construct occurred at a similar rate irerspective of theophylline concentration. This indicates that further optimization of the cleavage conditions must be undertaken to perfect the performance of the theophylline aptazyme in vitro.

GENERATION OF RNA USING RIBOZYME AFFINITY PURIFICATION:

We then attempted to purify RNA utilizing our ribosome affinity purification strategy.

Two separate constructs were employed, one expressing MS2 coat-protein under control of a standard RBS, and one under control of a weak RBS. In this way, we hoped to determine the optimum amount of MS2 expression required for effective purification of our desired RNA species.

Uleth15_Low and high RBS constructs.JPG

Initially, our constructs were cloned into BL21(DE3) cells for overexpression of MS2 in both cases upon induction with IPTG.

As illustrated, the overexpression of the Strong RBS Ribozyme Affinity Purification was more successful in purifying large amounts of MS2 coat-protein. This is certainly an expected result, and exhibits that the constructs were behaving appropriately. However, we also wanted to see if this increased amount of protein corresponded to an increase in amount of purified RNA.

Upon purification from the above fractions, the resultant RNA was subjected to concentrations of theophylline ranging from 10uM to 1mM and the cleavage was analyzed.